Microscopy

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Microscopy
( `* }4 N% F( _" g  T9 MMicroscopy is the science of producing and observing images of objects that cannot be seen by the unaided eye. A microscope is an instrument which produces the image. The primary function of a microscope is to resolve, that is distinguish, two closely spaced objects as separate. The secondary function of a microscope is to magnify. Microscopy has developed into an exciting field with numerous applications in biology, geology, chemistry, physics, and technology.
The light microscope
- I3 B% J6 v- t1 H% m- ~The most common, inexpensive, and easy to use microscope is the light microscope, which produces a magnified image of the object by bending and focusing light rays. The light microscope uses a variety of glass lenses to produce a magnified image that is focused before the eye. The magnifying properties of a converging lens, like that which is used in a typical magnifying glass or camera. Light from the object is bent, or refracted, as it passes through the lens producing an image which is inverted and magnified. In the simplest compound microscope, two converging lenses are used. The image from the first lens (objective) becomes the object for the second lens (eyepiece). The final image is much larger than either lens could produce independently. With a little effort, you can reproduce this effect yourself by using two magnifying glasses.
The wavelength of visible light ultimately limits the resolving power of the light microscope. Therefore, two objects separated by distances significantly less than about 0.4 micrometers (the smallest wavelength of visible light) cannot be distinguished as separate. This is because the light microscope produces its images by reflecting from or transmitting visible light through a specimen. An analogy can be made to ocean waves at the beach, with wavelengths of a few meters. If two people were wading into the surf only a few inches apart (a separation much less than the wavelength of the ocean waves), it would be impossible to distinguish them as separate by analyzing the ocean waves that reflected from them. Despite these limitations, the resolution of the light microscope is sufficient to produce excellent images of many of the important cell structures and organelles, and consequently still has many applications, chiefly in biology.
History of light microscopy
Since the time of the Romans, it was realized that certain shapes of glass had properties that could magnify objects. By the year 1300, these early crude lenses were being used as corrective eyeglasses. It wasn't until the late 1500s, however, that the first compound microscopes were developed.
Robert Hooke (1635-1703) was the first to publish results on the microscopy of plants and animals. Using a simple two lens compound microscope, he was able to discern the cells in a thin section of cork. The most famous microbiologist was Antoni van Leeuwenhoek (1632-1723) who, using just a single lens microscope, was able to describe organisms and tissues, such as bacteria and red blood cells, which were previously not known to exist. In his lifetime, Leeuwenhoek built over 400 microscopes, each one specifically designed for one specimen only. The highest resolution he was able to achieve was about 2 micrometers.
  a( O  e! N% V% O# s: u: X- [By the mid-nineteenth century, significant improvements had been made in the light microscope design, mainly due to refinements in lens grinding techniques. However, most of these lens refinements were the result of trial and error rather than inspired through principlesPage 2553 | Top of Article

A scanning electron microscope at the Billancourt Centre, Boulogne, France. This system is used in the testing of Renault Sport's motor racing engines. The samples on screen are the fracture faces of components subjected to a tensile strength test. Photograph by Philippe Plailly. Eurelious/Science Photo Library, National Audubon Society Collection/Photo Researchers, Inc. Reproduced by permission.of physics. Ernst Abbé (1840-1905) was the first to apply physical principles to lens design. Combining glasses with different refracting powers into a single lens, he was able to reduce image distortion significantly. Despite these improvements, the ultimate resolution of the light microscope was still limited by the wavelength of light. To resolve finer detail, something with a smaller wavelength than light would have to be used.
Electron microscopy
In the mid-1920s, Louis de Broglie (1892-1966) suggested that electrons, as well as other particles, should exhibit wave like properties similar to light. Experiments on electron beams a few years later confirmed de Broglie's hypothesis. Electrons behave like waves. Of importance to microscopy was the fact that the wavelength of electrons is typically much smaller than the wavelength of light. Therefore, the limitation imposed on the light microscope of 0.4 micrometers could be significantly reduced by using a beam of electrons to illuminate the specimen. This fact was exploited in the 1930s in the development of the electron microscope.
There are two types of electron microscope, the transmission electron microscope (TEM) and the scanning electron microscope (SEM). The TEM transmits electrons through an extremely thin sample. The electrons scatter as they collide with the atoms in the sample and form an image on a photographic film below the sample. This process is similar to a medical x ray where x rays (very short wavelength light) are transmitted through the body and form an image on photographic film behind the body. By contrast, the SEM reflects a narrow beam of electrons off the surface of a sample and detects the reflected electrons. To image a certain area of the sample, the electron beam is scanned in a back and forth motion parallel to the sample surface, similar to the process of mowing a square section of lawn. The chief differences between the two microscopes are that the TEM gives a two-dimensional picture of the interior of the sample while the SEM gives a three-dimensional picture of the surface of the sample. Images produced by SEM are familiar to the public, as in television commercials showing pollen grains or dust mites.
  @1 ~, f, t: K) x$ K8 OFor the light microscope, light can be focused and bent using the refractive properties of glass lenses. ToPage 2554 | Top of Article
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A typical compound microscope. Carolina Biological Supply Company/Phototake NYC. Reproduced by permission.bend and focus beams of electrons, however, it is necessary to use magnetic fields. The magnetic lens which focuses the electrons works through the physical principle that a charged particle, such as an electron which has a negative charge, will experience a force when it is moving in a magnetic field. By positioning magnets properly along the electron beam, it is possible to bend the electrons in such a way as to produce a magnified image on a photographic film or a fluorescent screen. This same principle is used in a television set to focus electrons onto the television screen to give the appropriate images.
8 ~. X) v) {" ?4 Y& y# G. c' MElectron microscopes are complex and expensive. To use them effectively requires extensive training. They are rarely found outside the research laboratory. Sample preparation can be extremely time consuming. For the TEM, the sample must be ground extremely thin, less than 0.1 micrometer, so that the electrons will make it through the sample. For the SEM, the sample is usually coated with a thin layer of gold to increase its ability to reflect electrons. Therefore, in electron microscopy, the specimen can't be living. Today, the best TEMs can produce images of the atoms in the interior of a sample. This is a factor of a 1,000 better than the best light microscopes. The SEM, on the other hand, can typically distinguish objects about 100 atoms in size.
Scanning tunneling microscopy
In the early 1980s, a new technique in microscopy was developed which did not involve beams of electrons or light to produce an image. Instead, a small metal tip is scanned very close to the surface of a sample and a tiny electric current is measured as the tip passes over the atoms on the surface. The microscope that works in this manner is the scanning tunneling microscope (STM). When a metal tip is brought close to the sample surface, the electrons that surround the atoms on the surface can actually "tunnel through" the air gap and produce a current through the tip. This physical phenomenon is called tunneling and is one of the amazing results of quantum physics. If such phenomenon could occur with large objects, it would be possible for a baseball to tunnel through a brick wall with no damage to either. The current of electrons that tunnel through the air gap is very much dependent on the width of the gap and therefore the current will rise and fall in succession with the atoms on the surface. This current is then amplified and fed into a computer to produce a three dimensional image of the atoms on the surface.
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, x. u9 [/ K2 `4 f9 O9 Q5 m; qWithout the need for complicated magnetic lenses and electron beams, the STM is far less complex than the electron microscope. The tiny tunneling current can be simply amplified through electronic circuitry similar to that which is used in other electronic equipment, such as a stereo. In addition, the sample preparation is usually less tedious. Many samples can be imaged in air with essentially no preparation. For more sensitive samples which react with air, imaging is done in vacuum. A requirement for the STM is that the samples be electrically conducting, such as a metal.
& i' A. B9 d5 Y2 SRecent developments in microscopy
There have been numerous variations on the types of microscopy outlined so far. A sampling of these is: acoustic microscopy, which involves the reflection of sound waves off a specimen; x-ray microscopy, which involves the transmission of x rays through the specimen; near field optical microscopy, which involves shining light through a small opening smaller than the wavelength of light; and atomic force microscopy, which is similar to scanning tunneling microscopy but can be applied to materials that are not electrically conducting, such as quartz.

( v' r* ?  S& [1 D+ SAn atomic force micrograph of the surface of a thin copolymer film. The atomic force microscope (AFM) is capable of atomic-scale resolution, and works by drawing a very fine probe across the surface of the sample. The vertical motion of the probe is converted into electronic signals which are then processed to give the type of surface map seen here. The advantage the AFM has over the scanning tunneling microscope is that it works for samples that are not electrical conductors. Photograph by Philippe Plailly. National Audubon Society Collection/Photo Researchers, Inc. Reproduced by permission.
One of the most amazing recent developments in microscopy involves the manipulation of individual atoms. Through a novel application of the STM, scientists at IBM were able to arrange individual atoms on a surface and spell out the letters "IBM." This has opened up new directions in microscopy, where the microscope is both an instrument with which to observe and to interact with microscopic objects. Future trends in microscopy will most likely probe features within the atom.
) U2 e( l) C7 b5 [. m. KResources
2 F! ^* w$ n, g5 |) J2 CBooks
Burgess, Jeremy, Michael Marten, and Rosemary Taylor. Microcosmos. Cambridge: Cambridge University Press, 1987.
5 b9 Y% P& y- v7 d( TGiancoli, Douglas. Physics. Englewood Cliffs, NJ: Prentice Hall, 1995.
Slayter, Elizabeth and Henry. Light and Electron Microscopy. Cambridge: Cambridge University Press, 1992.
/ K0 k! X! Y3 ?: X# Q$ kPeriodicals
Eigler, D.M., and E.K. Schweizer. &quotositioning Single Atoms with a Scanning Tunneling Microscope." Nature 344 (1990): 524-526.
Taylor, D., Michel Nederlof, Frederick Lanni, and Alan Waggoner. "The New Vision of Light Microscopy." American Scientist 80 (1992): 322-335.